Nucleoside-phospholipid conjugate

ABSTRACT

Novel nucleoside-phospholipid conjugates of the formula ##STR1## wherein R 1  is C 14-24  long chain aliphatic acyl or C 1-24  aliphatic alkyl and N s  is a nucleoside residue; and pharmacologically acceptable salts thereof are prepared in a one-step reaction by reacting the corresponding nucleoside and glycerophospholipid derivative in the presence of phospholipase D-P.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel nucleoside-phospholipidconjugates and salts thereof. More particularly, the present inventionrelates to a nucleoside-phospholipid conjugate of the formula ##STR2##wherein R₁ is C₁₄₋₂₄ aliphatic acyl or C₁₋₂₄ aliphatic alkyl, R₂ isC₂₋₁₀ aliphatic acyl or C₁₋₂₄ aliphatic alkyl, and N_(s) is a nucleosideresidue, and salts thereof.

2. Description of the Prior Art

Nucleoside antitumor agents have been widely used as effectivechemotherapeutics for neoplastic cells. In their application asantitumor-chemotherapeutics, however, several problems have arisen. Forexample, in the mechanism for the activity of nucleoside anti-neoplasticagents, in vivo phosphorylation of the hydroxyl group at the nucleoside5' position is essential for antitumor activity; the agent is decomposedto an inactive substance by inactivation reactions such asphosphorolysis and deamination; the resistance of tumor cells toantitumor agents increases; and the agent is sometimes toxic to normalmitotic cells. Many kinds of nucleoside derivatives have beensynthesized to overcome the disadvantages of known nucleoside antitumoragents.

CDP-diacylglycerol is known to have an important role as an intermediatein the biosynthesis of glycerophospholipid in vivo. Its analogue,arabinosylcytosine-phospolipid conjugate, which has antitumor activity,has been chemically synthesized [Biochim. Biophys. Acta, 619, 619-631(1980), J. Med. Chem., 1982, 25, 1322-1329].

5-fluoro-2-deoxyuridine-phospholipid conjugate (Japan Unexam. Pat. Publ.No. 61-91195, No. 61-152694) and a process for manufacturingheterocyclic phospholipid conjugate by phospholipase DM (ibid. No.61-88890) have also been reported.

This prior art has a number of disadvantages. Synthesis of these knowncompounds is a complicated multistep process with quite low overallyield. Moreover, these synthetic compounds are only sparingly soluble inaqueous media, and are thus difficult to use as injectable preparations.

Furthermore, previously reported nucleoside-phospholipid conjugatescomprise an ester linkage of glyceride and a hydrophobic fatty acid in aphospholipid backbone, and hence are easily decomposed by phospholipaseA₁ or phospholipase A₂ in vivo. Stability of these compounds is alsounsatisfactory.

SUMMARY OF THE INVENTION

We have found that phospholipase D-P effectively catalyzes the transferreaction of the phosphatidyl residue from glycerophospholipid to theprimary hydroxyl group of a nucleoside. A heretofore unavailable varietyof nucleoside-phospholipid conjugates can thus readily be prepare.

We have also found that nucleoside-phospholipid conjugates of the aboveformula, wherein R₁ is C₁₄₋₂₄ long-chain aliphatic acyl, R₂ is C₂₋₁₀aliphatic acyl or aliphatic alkyl and N_(s) is a nucleoside residue, andsalts thereof, have superior solubility in aqueous media and have strongantitumor activity.

We have further found that ether type compounds such asnucleoside-dialkylether phospholipid conjugates or salts thereof are notdecomposed by phospholipase A₁ or A₂ in vivo, and have strong antitumoractivity.

An object of the present invention is to provide novelnucleoside-phospholipid conjugates of the formula ##STR3## wherein R₁,R₂ and N_(s) have the same meanings as before.

The term "alkyl" as used in the present specification means saturatedalkyl or unsaturated alkyl such as alkenyl, alkadienyl or alkynyl.

Specifically, the group R₁ is C₁₄₋₂₄ long-chain aliphatic acyl or C₁₋₂₄aliphatic alkyl. The C₁₄₋₂₄ long-chain aliphatic acyl may be saturatedor unsaturated. Examples of straight chain saturated aliphatic acyl aremyristoyl, palmitoyl, stearoyl, eicosanoyl, dosaconoyl andtetracosanoyl, and examples of unsaturated aliphatic acyl arepalmito-oleoyl, oleoyl and 9,12-octa-decadienoyl. The C₁₋₂₄ aliphaticalkyl may also be saturated or unsaturated, and it may be straight orbranched. Examples of C₁₋₂₄ saturated aliphatic alkyl are methyl, ethyl,propyl, butyl, hexyl, heptyl, 3-heptyl, octyl, nonyl, decyl,8-ethyldecyl, undecyl, lauryl, tridecyl, pentadiecyl, hexadecyl (cetyl),heptadecyl, octadecyl (stearyl), nonadecyl nonadecynol and eicodecyl,and examples of C₂₋₂₄ unsaturated aliphatic alkyl are vinyl, allyl,2-butenyl, 3-hexynyl, 4-decynyl, 6-tetradecenyl, 9-octadecenyl andlinoleyl.

The group R₂ is C₂₋₁₀ aliphatic acyl or C₁₋₂₄ aliphatic alkyl. Examplesof C₂₋₁₀ aliphatic acyl are acetyl, propynoyl, propanoyl, butanoyl,hexanoyl, octanoyl and decanoyl. Examples of C₁₋₂₄ aliphatic alkyl arethe same as those for group R₁.

In the above compound, at least one of R₁ and R₂ is preferably C₁₄₋₂₄aliphatic alkyl, for affinity with the cell membrane in vivo.

The group N_(s) is a nucleoside residue, and is preferably aribonucleoside residue such as 5-fluorouridine-5'-yl, bredinine-5'-yl,tubercidin-5'-yl and 5-fluorocytidine-5'-yl, an arabinosyl nucleosideresidue such as arabinosyl cytosine-5'-yl,arabinosyl-5-fluorocytosine-5'-yl, arabinosyladenine-5'-yl andarabinosylthymine-5'-yl, a 2'-deoxyribonucleoside residue such as5-fluoro-2'-deoxyuridine-5'-yl, or a carbocyclic nucleoside residue suchas neplanocin A-6'-yl.

Salts of the above compounds can be pharmacologically acceptable saltssuch as alkaline metal salts, alkaline earth metal salts, transitionmetal salts and organic base salts.

A nucleoside-phospholipid conjugate according to the present invention,and salts thereof, can be obtained by reacting a glycerophospholipidderivative of the formula ##STR4## wherein R₁ and R₂ have the samemeanings as before, with a nucleoside of the formula N_(s) -OH, forexample a ribonucleoside such as 5-fluorouridine, bredinine, tubercidinor 5-fluorocytidine, or an arabinosyl nucleoside such as arabinosylcytosine, arabinosyl-5-fluorocytosine, arabinosyladenine or arabinosylthymine or a 2'-deoxyribonucleoside such as 5-fluoro-2'-deoxyuridine, ora carbocyclic nucleoside such as neplanocin A, in the presence ofphospholipase D-P in a suitable solvent.

A preferred example of phospholipase D-P is phospholipase D-P obtainedby culturing a broth of Streptomyces sp. AA586 FERM P-6100 (Japan Pat.Unexam. Publ. No. 58-152481, Toyo Jozo Co., Catalog No. P-39). Theamount of catalyst is at least 0.01 unit phospholipase D-P per 0.001mole of phospholipid, and is preferably 1-100 units. Examples of asuitable solvent are two-phase systems of organic solvent and aqueoussolvent, for example a mixture of organic solvent such as ether, benzeneor chloroform and buffer solution of pH 3-9, preferably pH 4-6. Ageneral example of a water soluble salt for generation of a metal ion iscalcium chloride. Reaction temperature is generally 20°-60° C. andreaction time is 30 minutes to 30 hours, preferably 1-6 hours. Thethus-obtained nucleoside-phospholipid conjugate according to theinvention can be purified by a partition method and silica-gelchromatography.

One-step synthesis of nucleoside-phospholipid conjugate of the presentinvention is illustrated as follows: ##STR5## wherein R₁, R₂ and N_(s)have the same meanings as before.

The thus-obtained product can be prepared as a non-toxic,pharmacologically acceptable salt, such as a sodium or potassium salt.

The thus-prepared nucleoside-phospholipid conjugate of the presentinvention has the advantages that: it is more lipophilic as comparedwith the original nucleoside; it is not easily excreted, that is, it ismore active for a longer time; it is not affected by enzymaticinactivation reactions such as phosphorolysis, deamination andreduction; it has higher affinity for cell membranes; antineoplasticnucleoside 5'-monophosphate is generated in cells without action ofkinase; and it has long action and increased activity with low toxicity.

The ester type nucleoside-phospholipid conjugate of the presentinvention is especially resistant to hydrolysis by phospholipase A₁ orphospholipase A₂ in vivo, and can be administered orally. Other types ofnucleoside-phospholipid conjugates according to the present inventionhave the advantage of high solubility in water, and hence they can beprescribed not only in oral administration form but also as aninjectable preparation.

The novel nucleoside-phospholipid conjugates of the present inventionreveal marked antitumor activity in vivo, in addition to anti-metastaticactivity on tumors, and anti-viral activity.

Antitumor activity against mouse leukemia P-388 carcinoma is shown inthe ensuring examples, according to the following list ofspecifications:

Antitumor Activity

1. Samples: shown in Tables 1 and 2.

2. Animal: BDF₁ mice, age 5 weeks, male, Charles River Inc., 5 mice inan experimental group of 7 control mice.

3. Tumor cells: P-388 leukemic cells; 1×10⁶ /0.2 ml are inoculatedintraperitoneally in BDF₁ mice of the experimental group.

4. Preparation of samples and administration of drugs:

Samples are suspended in Tris-HCl saline solution by sonification. 0.1ml/10 g body weight is administered.

Administration: starting one day after inoculation of tumor cells, oncea day for 5 days intraperitoneally. Amount of dose is shown in theTables.

5. Increase in life span (ILS) is calculated by the following equation:##EQU1##

Average survival data in control group is 7.86 days (7.57-7.93 days).

As shown in Table 1, each of the nucleoside-phospholipid conjugatesillustrated in this table is administered at 3-10 mg/kg/day in BDF₁ miceof the experimental group, and each is shown to have strong antitumoractivity, promoting an increased life span of 30-120%.

Also, as shown in Table 2, ester type nucleoside-phospholipid conjugatesillustrated in this table is administered at 10-30 mg/kg/day in BDF₁mice, and each is shown to have strong antitumor activity, promoting anincreased life span of 30-150%.

                  TABLE 1                                                         ______________________________________                                        nucleoside-phospholipid                                                       conjugate              dose     ILS                                           R.sub.1    R.sub.2  N.sub.5    (mg/kg)                                                                              (%)                                     ______________________________________                                        palmitoyl  decanoyl FUR        3 × 5                                                                          61.2                                                                   10 × 5                                                                         121.6                                   palmitoyl  octanoyl FUR        3 × 5                                                                          45.3                                                                   10 × 5                                                                         79.7                                    palmitoyl  hexanoyl FUR        3 × 5                                                                          45.3                                                                   10 × 5                                                                         79.7                                    palmitoyl  butanoyl FUR        3 × 5                                                                          36.1                                                                   10 × 5                                                                         53.2                                                                   3 × 5                                                                          40.1                                    palmitoyl  acetyl   FUR        10 × 5                                                                         58.9                                    palmitoyl  decanoyl NEPA       3 × 5                                                                          50.6                                                                   10 × 5                                                                         89.4                                    palmitoyl  octanoyl NEPA       3 × 5                                                                          66.4                                                                   10 × 5                                                                         84.9                                    palmitoyl  hexanoyl NEPA       3 × 5                                                                          34.7                                                                   10 × 5                                                                         53.2                                    palmitoyl  butanoyl NEPA       3 × 5                                                                          48.0                                                                   10 × 5                                                                         63.4                                    palmitoyl  acetyl   NEPA       3 × 5                                                                          37.5                                                                   10 × 5                                                                          55.1                                   ______________________________________                                         FUR: 5fluorouridine 5yl                                                       NEPA: neplanocin A 6yl                                                   

                  TABLE 2                                                         ______________________________________                                        Ester type nucleoside-                                                        phospholipid conjugate                                                                              dose     ILS                                            R.sub.1   R.sub.2   N.sub.5   (mg/kg)                                                                              (%)                                      ______________________________________                                        cetyl     cetyl     FUR       10 × 5                                                                         101.0                                                                  30 × 5                                                                         127.0                                    stearyl   stearyl   FUR       30 × 5                                                                         31.0                                     oleyl     oleyl     FUR       10 × 5                                                                         74.0                                                                   30 × 5                                                                         76.0                                     cetyl     cetyl     NepA      30 × 5                                                                         34.9                                     stearyl   stearyl   NepA      30 × 5                                                                         31.0                                     oleyl     oleyl     NepA      30 × 5                                                                         26.0                                     cetyl     cetyl     AraFC     10 × 5                                                                         68.0                                                                   30 × 5                                                                         151.9                                    stearyl   stearyl   AraFC     30 × 5                                                                         61.0                                     oleyl     oleeyl    AraFC     10 × 5                                                                         81.0                                                                   30 × 5                                                                         148.0                                    ______________________________________                                         FUR : 5fluorouridine-5yl                                                      NePA : neplanocin A  6yl                                                      AraFC : arabinosyl  5  fluorocytosine  5 yl                              

Acute Toxicity

The samples of Tables 1 and 2, suspended in Tris-HCl saline solution bysonification, are administered intraperitoneally at doses of 100 mg/kgfor the compounds of Table 1 and 200 mg/kg for the compounds of Table 2.No signs of toxicity were observed.

The nucleoside-phospholipid conjugates of the present invention andsalts thereof can be admixed with pharmacologically acceptableexcipients in carrier or diluent form, and administered as tablets,capsules, granules, powders, injectable mixtures or suppositories,either orally or parenterally.

The daily dosage amount may be 1-500 mg for humans, with one or moredoses per day; however, dosage will vary depending on the age, bodyweight, symptoms and condition of the patient.

The following examples illustrate the present invention but are not tobe construed as limiting:

EXAMPLES 1-12

Nucleoside N_(s) -OH (amount shown in Table 3) was dissolved in 200 mMacetate buffer pH 6.0 (volume shown in Table 3) containing 250 mM CaCl₂,and the resultant solution was stirred at 45° C. for 3 mins.Phospholipase D-P (3 mg, from Streptomyces, Toyo Jozo Co.) and a 0.3 mMchloroform solution (10 ml) of the phospholipid derivative indicated inTable 3 were added thereto, with the resultant mixture being stirred for6 hours and then cooled. To the reaction mixture were added 1N-HCl (5ml), chloroform (30 ml) and methanol (25 ml), and the resultant mixturewas then partitioned. The organic layer was washed twice with water anddried in vacuo. The residue was dried in vacuo after adding a smallamount of ethanol, and was then dissolved in a small amount ofchloroform, whereafter it was charged on a flash column of silica gel(2.5×10 cm) and eluted stepwise with chloroform, followed by a series ofchloroform:methanol mixtures of relative concentrations (20:1), (15:1),(12:1), (10:1), (7:1), (5:1), (4:1) and (2:1) in this order. The eluatewas dried in vacuo and the resultant residue was dissolved in a mixtureof chloroform (40 ml) and methanol (20 ml), and separated by adding0.5N-HCl (12 ml), then washed twice with water and dried to yield theproduct.

                                      TABLE 3                                     __________________________________________________________________________    No R.sub.1                                                                            R.sub.2                                                                            N.sub.5 --OH                                                                         Buffer solu.                                                                        Yield                                                                             PLDP                                                                              UV λmax                                                                      FAB mass spectrum                                                                        Rf                         __________________________________________________________________________    1  Palmitoyl                                                                          decanoyl                                                                           FUR, 10 eq.                                                                          B', 3 ml                                                                            59% 3 mg                                                                              268 nm                                                                              831 (M + Na)                                                                             0.32                       2  "    "    NepA, 5 eq.                                                                          B', 5 ml                                                                            63% 3 mg                                                                              260 nm                                                                              832 (M + Na)                                                                             0.28                       3  "    octanoyl                                                                           FUR, 10 eq.                                                                          B', 3 ml                                                                            58% 3 mg                                                                              268 nm                                                                              825 (M + 2Na - H)                                                                        0.31                       4  "    "    NepA, 5 eq.                                                                          B', 5 ml                                                                            61% 3 mg                                                                              260 nm                                                                              826 (M + 2Na - H)                                                                        0.25                       5  "    hexanoyl                                                                           FUR, 10 eq.                                                                          B', 3 ml                                                                            69% 3 mg                                                                              268 nm                                                                              797 (M + 2Na - H)                                                                        0.29                       6  "    "    NepA, 5 eq.                                                                          B', 5 ml                                                                            73% 3 mg                                                                              260 nm                                                                              776 (M + Na)                                                                             0.23                       7  "    butanoyl                                                                           FUR, 10 eq.                                                                          B', 3 ml                                                                            68% 3 mg                                                                              268 nm                                                                              769 (M + 2Na - H)                                                                        0.26                       8  "    "    NepA, 5 eq.                                                                          B', 5 ml                                                                            72% 3 mg                                                                              260 nm                                                                              748 (M + Na)                                                                             0.22                       9  "    acetyl                                                                             FUR, 10 eq.                                                                          B', 3 ml                                                                            65% 3 mg                                                                              268 nm                                                                              741 (M + 2Na - H)                                                                        0.21                       10 "    "    NepA, 5 eq.                                                                          B', 5 ml                                                                            69% 3 mg                                                                              260 nm                                                                              742 (M + 2Na - H)                                                                        0.17                       11 stearoyl                                                                           octyl                                                                              NepA, 5 eq.                                                                          B', 5 ml                                                                            62% 3 mg                                                                              260 nm                                                                              818 (M + Na)                                                                             0.27                       12 "    "    FUR, 20 eq.                                                                          B', 5 ml                                                                            55% 3 mg                                                                              268 nm                                                                              719 (M + Na)                                                                             0.22                       __________________________________________________________________________     B.sub.1 : 200 mM acetate bufer (pH 6.0) containing 250 mM CaCl.sub.2          PLDP: Phospholipase DP (specific activity : 160 units/mg)                     UV: measured in chloroformmethanol (1:2)                                      Rf: developer:chloroform:methanol:water (65:25:30) Plate; Merck, Art 5715     Spot was detected by UV and molybdenblue reagent.                        

EXAMPLES 13-22

Nucleoside N_(s) -OH (amount shown in Table 4) was dissolved orsuspended in 200 mM acetate buffer pH 6.0 (volume shown in Table 4)containing 250 mM CaCl₂ and the resultant mixture was stirred at 45° C.for 3 minutes. Phospholipase D-P (amount shown in Table 4) and 20 ml ofa 0.3 mM chloroform solution of phospholipid derivative (shown in Table4) were added thereto and the resultant mixture was stirred for 6 hoursand then cooled. 1N-HCl (5 ml), chloroform (30 ml) and methanol (25 ml)were added thereto and the organic layer was separated. The organiclayer was washed twice with water and dried in vacuo. A small amount ofethanol was added to the residue and the resulting solution was dried invacuo. The residue was dissolved in a small amount of chloroform andcharged on a flash column of silica gel (2.5×10 cm), where it was elutedstepwise with chloroform followed by a series of mixtures ofchloroform:methanol (20:1), (15:1), (12:1), (10:1), (7:1), (5:1), (4:1)and (3:1), in this order. The eluate was dried in vacuo and the residuewas dissolved in a mixture of chloroform (40 ml) and methanol (20 ml).0.5N-HCl (12 ml) was then added to separate the organic layer, which waswashed twice with water. The residue was dried in vacuo to obtain thedesired product.

NMR (CDCl₃ :CD₃ OD=2:1):

1: ppm 7.85 (d, 1H, J_(6F) =6.3H), 5.89 (bs, 1H), 4.3-3.9 (m, 7H), 3.53(m, 7H), 1.55 (br, 4H), 1.26 (s, 52H), 0.88 (t, 6H)

2: ppm 8.23 (s, 1H), 8.19 (s, 1H), 5.89 (bs, 1H), 5.52 (m, 1H), 4.72 (m,3H), 4.28 (m, 1H), 4.02 (m, 2H), 3.59 (m, 7H), 1.55 (br, 4H), 1,27 (s,52H), 0.88 (t, 6H)

3: ppm 8.22 (d, 1H, J_(6F) =6.5H), 6.11 (bs, 1H), 4.3-4.0 (m, 7H), 3.53(m, 7H), 1.55 (br, 4H), 1.26 (s, 52H), 0.88 (t, 6H)

                                      TABLE 4                                     __________________________________________________________________________    No                                                                              R.sub.1                                                                             R.sub.2                                                                             N.sub.5 --OH                                                                          Buffer solu.                                                                        Yield                                                                             PLDP                                                                              UV λmax                                                                      FAB mass spectrum                                                                       Rf                        __________________________________________________________________________    1 cetyl cetyl FUR, 20 eq.                                                                           3 ml  60% 3 mg                                                                              268 nm                                                                              887 (M + NBa)                                                                           0.33                      2 "     "     NePA, 5 eq.                                                                           7 m   73% 3 mg                                                                              260 nm                                                                              866 (MH)  0.30                      3 "     "     AraFC, 10 eq.                                                                         3 ml  36% 3 mg                                                                              283 nm                                                                              886 (M + Na)                                                                            0.24                      4 9-octadec-                                                                          9-octadec-                                                                          FUR, 20 eq.                                                                           5 ml  62% 2 mg                                                                              268 nm                                                                              939 (M + Na)                                                                            0.39                        enyl  enyl                                                                  5 9-octadec-                                                                          9-octadec-                                                                          NepA, 5 eq.                                                                           7 ml  78% 2 mg                                                                              260 nm                                                                              940 (M + Na)                                                                            0.34                        enyl  enyl                                                                  6 stearyl                                                                             octyl NepA, 5 eq.                                                                           7 ml  59% 5 mg                                                                              260 nm                                                                              804 (M + Na)                                                                            0.25                      7 stearyl                                                                             methyl                                                                              FUR, 20 eq.                                                                           5 ml  51% 5 mg                                                                              268 nm                                                                              705 (M + Na)                                                                            0.21                      8 cetyl methyl                                                                              FUR, 20 eq.                                                                           5 ml  55% 5 mg                                                                              268 nm                                                                              677 (M +  Na)                                                                           0.20                      9 cetyl hexyl FUR, 20 eq.                                                                           5 ml  61% 5 mg                                                                              268 nm                                                                              747 (M + Na)                                                                            0.25                      __________________________________________________________________________     Abbreviations are the same as in Table 3                                 

What is claimed is:
 1. A compound of the formula: ##STR6## wherein R₁ isC₁₄₋₂₄ long chain aliphatic acyl, R₂ is C₂₋₁₀ aliphatic acyl and N_(s)is neplanocin A-6'-yl, and pharmacologically acceptable salts thereof.2. Compound according to claim 1 wherein R₁ is C₁₆₋₁₈ long chainaliphatic acyl.